Dopaminergic neurons in the midbrain control aspects of mood, cognition, and movement and are involved in the pathogenesis of a variety of disorders including drug addiction. A primary regulator of dopaminergic neurotransmission is the dopamine transporter, which is responsible for the re-uptake of dopamine back into the pre-synaptic neuron. Drugs of addiction, such as amphetamine, disrupt dopamine re-uptake not only via competitive inhibition of transport but also by harnessing the neuron's endogenous signaling and endocytic pathways to down-regulate transporter expression at the cell surface. Previous research has implicated a number of signaling and endocytic pathways in the regulation of dopamine transporter surface expression. For example, activation of protein kinase C by its potent activator phorbol ester has been observed to increase transporter internalization in cell lines and rodent neurons. The critical barrier in transforming the identification of protein kinase C as a relevant signaling pathway into a therapeutic and/or diagnostic target is the lack of mechanistic insight into how protein kinase C regulates dopamine transporter trafficking in dopamine neurons. While PKC-stimulated DAT endocytosis is known to be dependent on DAT ubiquitination, few mechanistic details have been revealed, such as the isoform of PKC involved or what endogenous brain compounds stimulate PKC-mediated endocytosis. To address this barrier to progress, I will examine mechanisms of dopamine transporter regulation specifically in dopamine neurons to clarify the isoform of protein kinase C that regulates transporter endocytosis and ubiquitination. Preliminary studies indicate an important role for protein kinase C-epsilon, an isoform expressed in dopamine neurons, prompting me to propose the hypothesis that protein kinase C-epsilon regulates dopamine transporter endocytosis in dopaminergic neurons. I will test this hypothesis by using traditional neurochemical as well as innovative approaches. For example, I will control gene function selectively in dopaminergic neurons of dopamine transporter-Cre mutant mice by stereotaxic delivery of viral vectors that contain a Lox-stop-Lox transcriptional stop cassette. Additionally, I will complement our studies of endogenous transporter trafficking by analyzing the endocytosis of an extracellular epitope-tagged mutant transporter exogenously expressed in mouse dopamine neurons. I expect that the results will lay the foundation for future mechanistic and behavioral experimentation that may eventually lead to the identification of new therapeutic targets for modulating pathological dopaminergic signaling.